Identification of an exogenous intra-erythrocytic bacterium in patients having systemic lupus erythematosus, and treatment

ABSTRACT

Intra-erythrocytic exogenous bacterial structures or parasites seen by giemsa and phase contrast microscopy in several patients with systemic lupus erythematosus (SLE) and not in controls were identified as bacteria. Treatment of an SLE patient is contemplated with an antibacterial amount of a rifamycin in conjunction with an antibacterial amount of either a macrolide such as clarithromycin or a third generation cephalosporin such as cefpodoxime that is itself more preferably used in conjunction with an adjuvant amount of probenecid.

This is a division of application Ser. No. 08/763,709, now U.S. Pat. No.5,795,563, filed Dec. 13, 1996, which is a continuation-in-part ofapplication Ser. No. 08/707,472, filed Sep. 4, 1996, abandoned, whosedisclosures are incorporated by reference.

TECHNICAL FIELD

The present invention relates to identification of the presence of anexogenous structure within human erythrocytes or bone marrow cells andtreatment thereof, and more particularly to an assay for the presence ofan exogenous bacterial structure or parasite within the erythrocytes ofpatients having systemic lupus erythematosus (SLE) and treatment of suchpatients to lessen or eliminate the erythrocytic or bone marrow load ofthose bacterial structures.

BACKGROUND OF THE INVENTION

Systemic lupus erythematosus (SLE) is a protean disease of unknownetiology which affects multiple organs. Lahita, R. G. Systemic LupusErythematosus, Churchill, Livingston, N.Y. (1987) page XXIX. Althoughthere is a marked similarity to an infectious entity, an exhaustivesearch for an etiologic agent has not yielded any proven candidates thatfulfill the criteria for causation of this disease. Crow et al.,"Etiologic Hypothesis for Systemic Lupus Erythematosus," in LahitaSystemic Lupus Erythematosus, Churchill, Livingston, N.Y. (1987) page 51ff. There is general agreement that tissue and organ injury in SLE ismediated by immune phenomena. Unexplained at this time is thepredilection of SLE for females. Taurog et al., Intern. J. Derm.,20:149-158 (1981).

Early descriptions of SLE before the advent of suppressive therapysuggested sepsis, and included fever, striking constitutional symptomsand high mortality. Crow et al., "Etiologic Hypothesis for SystemicLupus Erythematosus," in Lahita Systemic Lupus Erythematosus, Churchill,Livingston, N.Y. (1987) page 54. Recently, many viral etiologic agentshave been sought; none have been convincingly demonstrated. Pincus,Arthr & Rheum, 20:149-158 (1982). More recently the characterization ofsoluble products of bacteria and mycoplasmas with unique capacities toperturb immune systems have led to new considerations in regard to theinfectious trigger of SLE.

Intra-erythrocyte organisms with characteristics like theAnaplasmataceae that were thought to be Haemobartonella-like were firstsuggested as exciting exogenous agents in SLE by Kallick et al., NatureNew Biology, 236:145-146 (1972). The Anaplasmataceae family of bacteriaare Proteobacteria of the order Rickettsiales. That report was furtherdeveloped by a later report of antigenic similarities between SLE orlupus nephritis and diseases caused by Anaplasma marginale, anintra-erythrocytic parasite of cattle, and a member of the familyAnaplasmataceae. Kallick et al., Arthr. Rheum., 23:197-205 (1980).

Exogenous intra-erythrocytic structures seen in the same erythrocyte bygiemsa staining and phase contrast microscopy, which were identical orvery similar in appearance to Anaplasma marginale, have been observed inmost patients with SLE. However, because the number of such structuresseen in erythrocytes of SLE patients is very small, their presence hasbeen unconfirmed by electron microscopy, until the present study. Forexample, the number of infected erythrocytes is usually less than 0.1percent of observed erythrocytes in fixed thin blood films.

Based upon available data, which describe the pathology of SLE as tissuedestruction associated with antibody formation and the development ofantibodies against many body tissues as well as pathogens such asEpstein-Barr virus, and assuming that Anaplasmataceae or similarintra-erythrocytic pathogens are present in an uncontrolled parasitemiain SLE, a pathological mechanism can be deduced that contains, at leastin part, a continued antigenic stimulation of the B-cell immunemechanism. The Anaplasmataceae were thought to offer an ideal putativemodel of infection, in that, based on studies by the inventor, above,they present an approximately 1 percent parasitemia or less level insystemic lupus erythematosus patients.

The Anaplasmataceae have been extensively studied in animals, andalthough they affect erythrocytes as a primary organ, their pathogenicpotential appears to be primarily or exclusively mediated by immunemechanisms primarily identified as humoral. Immunity to Blood parasitesin Animals and Man, Miller et al. eds., Plenum Press, New York (1977)pages 155-188. In animal disease, antibiotics control the primarypathologic event, hemolytic anemia, only if given early in the course ofthe illness. Late in the course of animal illness the hemolytic anemiais mediated by antibody formation and antibiotics do not appear toaffect the course of the usually studied hemolytic anemia.

In animals, arthritis and long-term effects of illness after infectionhave not been studied or reported. However, in some animals that sufferhemolytic anemia such as adult bulls, chronic arthritis and anemia havebeen reported. In the veterinary literature, an antibiotic that appearsto suppress some of the clinical manifestations of these organisms istetracycline or one of its analogues. Franklin et al., SouthwesternVet., 15:131-139 (1962).

Several humans with SLE or connective tissue disease have been treatedwith tetracycline (doxycycline) in preliminary work of the inventorbased on the presumption of Anaplasmataceae parasitemia. Three patientsare exemplary.

The first was a 17 year old female with severe SLE and nephritis whoexperienced a lysis of fever within a week of therapy with disappearanceof Haemobartonella-like agents from the circulating erythrocytes asobserved by acridine orange and fluorescent antibody determination. Thispatient was not subsequently followed.

The second patient is a male with SLE who has been taking tetracyclinefor his lupus for 10 years. He stated that his fever, joint pains, andother symptoms disappeared while he was taking tetracycline. He hadfirst been given the antibiotic for treatment of another infection andnoted it caused amelioration of his SLE.

The third is a patient who has mixed connective tissue disorderresembling SLE but with a negative ANA titer. This patient went intoremission of her symptomatology after 3 weeks of therapy withtetracycline and has remained in clinical remission for the subsequent 3months. It is of interest that in addition to marked subjectiveimprovement of this last patient, C-reactive protein became negativeafter tetracycline therapy was begun.

Subsequent to these studied patients, a large number of other patientswith SLE have received chronic therapy with tetracycline or itsderivatives. These treatments have been on a compassionate basis by thepatients' own physicians, or as part of a study approved by aninstitutional review board, but not completed. Most of such treatmentshave resulted in amelioration of the disease state, with completeremission, or a trend in such amelioration. That study, done at CookCounty Hospital, Chicago, Ill., was terminated before the results, asanalyzed, were shown to be statistically significant. The resultsappeared to be based on the small numbers analyzed.

A current patient studied is a splenectomized female with SLE who had alarge number of parasitized erythrocytes beginning with about 16percent. She has been on doxycycline with informed consent for 16 monthswith initial and continued improvement. Because she has enoughintra-erythrocytic parasites to be counted, her course of treatment hasbeen followed and electron microscopy was carried out. When examined inDecember 1995, she still had about 1.1 percent parasitized erythrocytesseen by giemsa staining and phase contrast microscopy.

Aureomycin, a tetracycline-like drug, has been proposed as a treatmentfor rheumatoid diseases in the past by T. Brown and co-workers andothers, who treated a variety of patients with arthritis and lupus withclaims of some degree of success in the 1940's. [Brown et al., J. Lab.Clin. Med., 34:1404-1410 (1949); Scheff et al., Infec. Dis., 98:113(1956)] Those studies were not controlled, and have not been repeated.These theoretical and observed phenomena suggest that tetracycline maybe of benefit in the syndrome of SLE.

Present therapy of SLE is based upon heavy steroid use withimmunosuppressives and/or plasmapheresis. It is of interest thatAnaplasmataceae infections in animals are, almost uniquely amonginfectious diseases, ameliorated by steroids.[Scheff et al., Infec.Dis., 98:113 (1956); Ristic et al., J. Vet. Res., 19:37 (1958)]

No presently used therapy is completely satisfactory, and, although thelife expectancy of lupus patients has been considerably increased bypresent therapy, the ravages of therapeutic side effects and theconstant fatigue takes a severe toll in well being, general health, andincreased morbidity and mortality of the estimated 500,000 Americanswith this disease. Dubois, Lupus Erythematosus, 2d ed., U.S. CaliforniaPress, Los Angeles (1974).

The spleen is regulatory in removing nuclear remnants and otherintra-erythrocytic particles from erythrocytes, and in Anaplasmosis aswell as malaria, splenectomy causes a recrudescence of clinical diseaseand parasitemia. Kreier, Ann. Rev. Microbiol., 325-38 (1981).Splenectomy is an infrequently used treatment for the thrombocytopeniaseen in some patients with SLE. Coon, So. Amer. J. Surgery, 155:391(1988). However, the one splenectomy patient noted before was found tohave parasitemias of erythrocytes with intra-erythrocytic phasecontrast-visible refractile bodies in up to about 16 percent of herstudied erythrocytes (FIGS. 1 and 2). These intra-erythrocytic bodieswere very similar or identical to those seen in bovine A. marginaleinfection.

It would therefore be beneficial if the stainable exogenous structuresfound in the erythrocytes of SLE patients could be accuratelyidentified, readily assayed for and eliminated from a patient's blood inthat it is believed that those exogenous structures are the active,causative agent for SLE. The description that follows describes atreatment regimen that eliminates those exogenous bacterial structuresfrom an SLE patient's blood.

In 1891 Howell described intra-erythrocytic bodies that occurred insplenectomized patients and animals. The appearance of these bodies thatnow bear the name Howell-Jolly bodies followed within a few hours ordays of splenectomy. The elegant drawings of Dr. Howell, whose findingshave been subsequently confirmed by others with modern methods includingelectron microscopy, have amptly demonstrated this phenomenon. Thespleen in the normal patient removes the few nuclear remnants that arenot absorbed or extruded in the bone marrow.

Howell-Jolly bodies are described as about 1 μ in diameter in aneccentric position in the erythrocyte. The bodies seen in anaplasmosisand in the patients with SLE are approximately 0.5 μ and have phaserefractile characteristics that are not described in Howell-Jollybodies. It is believed that the site of generation of the agents seen inSLE patients'erythrocytes is in the normoblasts or other cells of thebone marrow. These agents are normally removed by the spleen, but in thesplenectomized patient are constantly circulating until the erythrocyteis senescent and destroyed in the reticulo-endothelial system. Theseparticles in the normoblasts appear to have heretofore escaped detectionbecause they morphologically resemble the normal metamorphosis of thenormoblast, but are smaller.

BRIEF SUMMARY OF THE INVENTION

It has now been found that the giemsa- and acridine orange-stainable andphase contrast microscopy visible exogenous structures or parasitesfound in the erythrocytes of SLE patients are neither Rickettsiae norAnaplasmatacea, but may be a presently unidentified member of theα-purple bacteria or another previously unclassified agent. Inaccordance with that finding, a process for treating SLE patients toremove the erythrocyte-parasitizing exogenous bacterial structures hasbeen found.

Thus, this invention contemplates a process for treating a patient whoseerythrocytes contain exogenous structures stainable with acridine orangeor giemsa and are visible in phase contrast microscopy, such as apatient having systemic lupus erythematosus. That process comprisesadministering to the patient an antibacterial amount of a thirdgeneration cephalosporin or a macrolide in connection with anantibacterial amount of a rifamycin. In preferred practice, an adjuvantamount of probenecid is administered in conjunction with theadministration of the third generation cephalosporin.

Most preferably, all three medicaments are administered in conjunctionwith each other when the cephalosporin is used. The administration ofthe macrolide or third generation cephalosporin plus a rifamycin ispreferably repeated periodically. Clarithromycin is a particularlypreferred macrolide, whereas cefpodoxime proxetil, a prodrug form ofcefpodoxime, is a particularly preferred third generation cephalosporin,and rifampin or rifabutin is the preferred rifamycin.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a part of this invention:

FIG. 1 is a thin blood film from the patient of the Example stained forone hour with filtered giemsa stain. This patient was splenectomized andhad systemic lupus erythematosus. Several individual erythrocytes arevisualized. The stained structure within the erythrocyte indicated bythe arrow is the unidentified infectious bacterium. The structure inanother erythrocyte designated "H" is a Howell-Jolly body often seen insplenectomized patients. A Howell-Jolly body can be differentiated fromthe bacterium by size and phase refraction but not by stainingcharacteristics. Original magnification ×630.

FIG. 2 is the same field of erythrocytes of the patient as seen inFIG. 1. The same erythrocytes are visualized, but the optical view is byphase contrast. In this mode, the giemsa-stained bodies are poorlyvisualized. Doubly refractile structures occupy the same positions andlocations within the erythrocytes of those structures designated as theunidentified infectious bacteria, as seen with phase contrast optics.The structure of FIG. 1 designated "H" is absent in the phase contrastview as the Howell-Jolly structure does not contain a refractile body.Original magnification is ×630.

FIG. 3 is an electron micrograph showing a small portion of anerythrocyte from the same patient as in FIGS. 1 and 2. The areadesignated "E" is the matrix of the erythrocyte. The small arrow aboveand to the right of the "E" designates the erythrocyte membrane. Thelarge arrow designates the double membrane enclosing the exogenous,infectious bacterium, and appears to have some of the characteristics ofa gram-negative bacterial membrane. The limiting membrane of the vacuolelayer surrounding the bacterium is designated by the unmarked wedge nearthe left-hand side of the micrograph, and morphologically resembleserythrocyte membrane. The original magnification ×65,000.

The present invention has several benefits and advantages.

One benefit of the present invention is that it provides a process fortreating a patient with SLE to lessen or remove the etiological bacteriafrom the erythrocytes of an SLE patient.

An advantage of the invention is that its process of treatment can becarried out with an antibacterial agent whose safety in humans hasalready been demonstrated.

Still further benefits and advantages of the present invention will beapparent to a skilled worker from the description that follows.

DETAILED DESCRIPTION OF THE INVENTION

A 30 year old African American female patient who had beensplenectomized presented with the symptoms of systemic lupuserythematosus (SLE) in 1993. Examination of her blood indicated thatabout 16 percent of her erythrocytes contained exogenous bacterialstructures or parasites that were stainable with giemsa and acridineorange and were refractile in phase contrast microscopy. Similarexogenous bacterial structures or parasites have also been found in theerythrocytes of other SLE patients, albeit at a lower level of infectioninvolving about 0.1 percent of the erythrocytes. A blood sample receivedfrom another splenectomized patient from Norway evidenced parasitizationof about 0.6 percent of the erythrocytes.

Inasmuch as the erythrocytes of the SLE patients examined have beenfound to contain these giemsa- and acridine orange-stainable exogenousbacterial structures or parasites, and such exogenous bacterialstructures are not present in the erythrocytes of healthy patients orpersons suffering from other diseases so far examined, it is believedthat those parasites are the infective agent that causes SLE.

In accordance with recognized methods of determining the presence of aninfectious agent in human specimens, a study was devised to assaywhether the same organism was identifiable in other patients with SLEand not in controls. Thus, blood was collected from patients with SLEand matched controls, and mixed with a small quantity of EDTA that hadbeen filtered through a 0.2 micron filter. Of the SLE patients studied,almost all exhibited the paratizization.

SLE affects every organ system and this is consistent with anintra-erythrocytic organism carried in a small number of erythrocytesthat may be capable of infecting cells other than erythrocytes. Afterprocessing in the reticulo-endothelial system of a patient with SLE,products from the breakdown of constantly circulating bacteria arecapable of inducing B cell stimulation, which can account for the highlevels of antibodies seen in SLE.

These organisms have now been found in the majority of patients withSLE, but have escaped detection and culturing by investigators over theyears. In almost all SLE patients examined, this new organismparasitizes less than one percent of erythrocytes, and the methodologyof examining human blood films is crude unless very careful technique isused. Both giemsa and Wright's stains must be filtered at each stainingor the precipitate can be confused with intra-erythrocytic bodies.

For example, stock giemsa stain was mixed with supplied buffer at aratio of 2 ml to 50 ml buffer. The stain was filtered onto thepreviously fixed thin blood film through a 0.2 micron filter. The stainmust not be allowed to stand for more than the time needed for staining.The slide was immersed in the diluted stain for one hour, after which itwas washed in de-ionized water and air dried.

The blood was examined on a Zeiss microscope through an objective withphase contrast optics. Each identified particle was viewed through ahigh power light field under oil immersion.

The exogenous intracellular structures of the unknown bacteria areobserved as blue gray bodies usually in the marginal position and areabout 0.4 to about 0.5 micron in longest dimension, whereas Howell-Jollybodies are larger and are not phase contrast retractile. (FIG. 1).Verification that the exogenous bacterial structures are not artifactsis made by switching to phase contrast microscopy without moving thestage. The blue grey structure under phase contrast appears as a doublyretractile structure in the same erythrocyte in the same location (FIG.2). These intra-erythrocyte structures can be also be confused withHeinz bodies, Howell-Jolly bodies (that are not retractile as is seen inFIG. 2), nuclear remnants, or simply be overlooked if not carefullysought.

Acridine orange can also be used as a stain for the exogenous bacterialstructures. In that case, the blood film is fixed with saline to which10 volume percent formalin has been added. After complete fixation for24 hours, the film is examined under indirect fluorescent microscopy.Intraerythrocytic structures containing RNA fluoresce bright orange andare usually present in the marginal position within an erythrocyte.

The quantification of percentage of erythrocytes parasitized was made bycounting ten fields within a square defined by an optically projectedprism, determining the number of parasitized erythrocytes within the tenfields, and dividing the total number of parasitized erythrocytes by thetotal number ×100. This product was identified as the percentage ofparasitized erythrocytes.

The standard method for staining of Anaplasma marginale using giemsastain was used here. One could not be sure of seeing an artifact unlessphase contrast microscopy was used to confirm each individual structureidentified. Only after dual viewing, was the observed blue grey bodyaccepted as a structure within the erythrocyte stroma.

Experimental infection of animals with Anaplasma is used for study ofthat entity, and veterinary hematologists have not relied on phasemicroscopy, because they see parasitemias of 50 to 80 percent. Someinvestigators in this field were unaware that the organisms could beseen more easily by phase contrast microscopy.

The presumed flooding of the reticuloendothelial system by thedegenerative products of the unidentified bacteria affords a readyexplanation for the spectrum of autoantibodies that characterize theextensively studied spectrum of B and T cell activation in SLE.

Treatment Process

The present invention contemplates treatment of a patient whoseerythrocytes contain exogenous bacterial structures that are stainablewith acridine orange and with giemsa, and are also visible with phasecontrast microscopy, such as a patient suffering from systemic lupuserythematosus (SLE). That treatment comprises administering to thatpatient an antibacterial amount of a rifamycin along with anantibacterial amount of a macrolide or a third generation cephalosporin.In more preferred practice, an adjuvant amount of probenecid isadministered in conjunction with the administration of the thirdgeneration cephalosporin.

SLE patients are often treated with a steroid compound such asprednisone as a palliative. That treatment is maintained during theantibacterial treatment and as long as is required.

Macrolides are hydroxylated macrocyclic lactones having 12 to 20 atomsin the primary ring. Although more than thirty macrolides are known,only erythromycin, its analogues and derivatives are clinicallyimportant, and the word macrolide is used herein to mean such compounds.A macrolide can be administered orally or parenterally, and again oraladministration is preferred.

As used herein, a derivative of erythromycin has a biological activitysimilar to that of erythromycin itself, and a macrocyclic ring structureand pendant saccharide moieties that are the same as erythromycin, butadditionally has one or more erythromycin substituent groups reactedwith another moiety to form a functional group different from thatpresent at the same position in erythromycin, such as one or moreerythromycin hydroxyls being etherified with a methyl group oresterified with an acetyl or propionyl group in the derivative.Exemplary erythromycin derivatives include clarithromycin (methylether), erythromycin 2'-acetate octadecanoate (2'-acetate, stearatesalt), erythromycin estolate (2'-propanoate dodecylsulfate salt) and thelike. Clarithromycin is particularly preferred.

An erythromycin analogue is a compound that has a biological activitysimilar to that of erythromycin itself, a macrocyclic ring structurethat is different from erythromycin and contains two atoms more or lessin the macrocyclic ring than does erythromycin, while having similarring substituents to erythromycin or an erythromycin derivative. Anexemplary erythromycin analogue is azithromycin.

Clarithromycin is available as BIAXIN™ FILMTAB® tablets from AbbottLaboratories. These commercial tablets each contain 250 mg or 500 mg ofclarithromycin. Usual dosages are 250 to about 4000 mg per day in one tofour about evenly spaced oral administrations. A daily administration ofa total of about 500 to 1000 mg in two to four administrations ispreferred with rifampin for synergetic activity.

Clarithromycin can be administered without regard to a patient's stomachcontents; i.e., given on a full or empty stomach. On the other hand, thebioavailability of erythromycin is lessened when that drug isadministered to a patient who had recently eaten.

Cephalosporin compounds are grouped in the art by so-called generationsthat are classed by activity rather than structure. Exemplary first,second and third generation classifications are found in Goodman andGilman's The Pharmaceutical Basis of Therapeutics, 8th ed., McGraw-Hill,Inc., New York (1993) pages 1085-1092; Remington's PharmaceuticalSciences, 18th ed., Gennaro et al. eds., Mack Publishing Co., Easton,Pa. (1990) pages 1193-1200; Physician's Desk Reference, PDR®, 48th ed.,Medical Economics Data Production Co., Montvale, N.J., Product CategoryIndex, (1994) pages 203-204, and individual listings; and The MerckIndex, 11th ed., Merck & Co., Rahway, N.J. (1989), individual listings.

Third generation cephalosporins are noted to be generally less activeagainst gram-positive organisms than are first generationcephalosporins, but are much more active against gram-negativeorganisms, particularly against Enterobacteriaceae.

One structural feature shared by many third generation cephalosporins isa second ring joined to the beta-lactam-sulfur-containing 4,6-fused ringthat usually characterizes cephalosporins, with that second ring beinglinked to the first ring by an alkoxyiminoacetyl amino group in whichthe alkoxy group is methyoxy or an --O--C₁ --C₄ --alkylenecarboxyl groupsuch as a --CH₂ CO₂ H or --C(CH₃)₂ CO₂ H group. It is noted, however,that cefuroxime, a second generation cephalosporin, also contains thatstructure, and that some third generation cephalosporins such ascefoperazone, cefpimizole and cefsulodin do not contain analkoxyiminoacetyl group. In addition, moxalactam, another thirdgeneration cephalosporin, has an oxygen rather than a sulfur atom in the4,6-fused ring. Thus, again, a third generation cephalosporin compounduseful herein is functionally defined as such in the art.

Exemplary third generation cephalosporin compounds useful in this aspectof the invention include cefoperazone, cefmenoxime, ceteferam,cefpimizole, cefodizime, cefixime, cefotaxime, cefsulodin, ceftazidime,ceftizoxime, ceftibuten, cefxiofur, ceftriaxone, cefuzonam, moxalactamand cefpodoxime. Most of the third generation cephalosporins areadministered by injection. However, oral administration is preferred.Orally active and administrable third generation cephalosporins includecefixime, cefteram, ceftibuten and cefpodoxime. Use of cefpodoxime isparticularly preferred, and that third generation cephalosporin isusually administered as an ester prodrug called cefpodoxime proxetil.This compound is available from The Upjohn Company as tablets or as anoral suspension under the name VANTINTM™.

The third generation cephalosporin, however administered, isadministered in an usual antibacterial amount. That amount differs witheach drug, with the usual antibacterial amounts being available in thePDR®. For the particular preferred cefpodoxime proxetil, that amount isabout 200 to about 800 milligrams (mg) and more preferably about 400 toabout 800 mg per day for adults. A usual duration of administration forthis third generation cephalosporin is 14 days, although that time maybe lengthened.

In preferred practice, the third generation cephalosporin isadministered in conjunction with the administration of an adjuvantamount of probenecid. Probenecid is available from Merck & Co. under thename BENEMID®, and is a well-known adjuvant for beta-lactam antibioticssuch as penicillins and cephalosporins, and acts to raise the bloodlevel of the beta-lactam by inhibiting excretion in urine.

Typical oral adult administrations are about 500 mg to about 2 grams (g)daily, more preferably about 250 to about 1000 mg daily, with a usualdose being 250 mg twice a day for one week, and then 500 mg twice a daythereafter. Probenecid and the third generation cephalosporin areusually administered at the same time. However, simultaneousadministration is not necessary so long as plasma levels of about 30 toabout 200 micrograms per milliliter (μg/mL) of probenecid in plasma areachieved when the third generation cephalosporin is in the bloodstream.Simultaneous oral administration is preferred.

It is necessary to co-administer a rifamycin with the other twomedicaments. The phrase "a rifamycin" is meant to include rifamycinitself as well as rifamycin derivatives as are discussed below.Rifamycin is a broad-spectrum antibiotic produced by Streptomycesmediterranei that is active against most gram-positive organisms and hasvariable activity against gram-negative organisms such as Escherichiacoli and Pseudomonas. Rifamycin and its derivatives also haveintracellular bactericidal activity.

Rifampin is a particularly preferred rifamycin derivative that isavailable from Marien Merrell Dow as RIFADIN®, and can administeredperorally or by injection in an antibacterial amount. Capsules for oraladministration are available that contain 150 or 300 mg of rifampin percapsule. Usual adult oral administrations are 600 mg once per day,usually with water about one hour before a meal, with dosages of about450 to about 900 mg per day being contemplated.

Rifabutin, available under the trademark MYCOBUTIN® fromPharmacia-Upjohn, is also a preferred rifamycin derivative. Rifaentine,available from Marion Merrell Dow under the trademark RIFATER®, andrifamide, also available from Marion Merrell Dow, are other rifamycinderivatives that can also be used.

One way to assess how long to continue administration of the macrolideor third generation cephalosporin in conjunction (along) with one orboth of the above drugs is to continue administration of the drugs untilthe stainable, phase contrast microscopically visible, exogenousbacterial structures seen in SLE patient's erythrocytes prior totreatment are absent from the erythrocytes. This duration ofadministration may take as long as about 120 days, the average lifetimeof an erythrocyte. Periodically repeated administrations of two or threedrugs that encompass the average lifetime of an erythrocyte are thuscontemplated and are preferred.

It is to be understood that the macrolide or third generationcephalosporin, probenecid and rifampin need not be administered via theabove-noted commercially available forms. Rather, those drugs can becompounded into a composition for administration to a SLE patient usingwell-known pharmaceutical techniques.

A contemplated composition can be a solid or a liquid. The activeingredients can also be individually admixed as a suspension of solidsin a solid or liquid physiologically tolerable carrier, or dissolved asa solute or suspended in the carrier, or a combination thereof.

Physiologically tolerable carriers are well-known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that can contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose and other solutes.These latter carriers are exemplified by Ringer's Injection, DextroseInjection, Dextrose and sodium chloride Injection and Lactated Ringer'sInjection.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as sesame oil or cottonseed oil, andwater-oil emulsions.

Exemplary solid carriers include those materials usually used in themanufacture of pills or tablets, and include corn starch, lactose,dicalcium phosphate, thickeners such as tragacanth and methylcelluloseU.S.P., finely divided SiO₂, polyvinylpyrrolidone, magnesium stearateand the like. Additionally, the solid carrier can include biodegradableand nonbiodegradable polymers, polypeptide carriers, affinity carrierssuch as AFFI-GEL® 601 (phenyl boronate resin available from Bio-RadLaboratories, Richmond, Calif.), liposomes and synthetic polymers, asare known in the art. Antioxidants such as methylparaben andpropylparaben can be present in both solid and liquid compositions, ascan sweeteners such as cane or beet sugar, sodium saccharin, sodiumcyclamate and the dipeptide methyl ester sweetener sold under thetrademark NUTRASWEET® (aspartame) by G. D. Searle Co.

It is particularly preferred, however, to utilize the orallyadministratable tablet and capsule forms of a macrolide such asclarithromycin as are available from the before-named manufacturers withrifampin or third generation cephalosporin, such as cefpodoximeproxetil, probenecid and rifampin.

EXAMPLE

The following example is presented to illustrate certain aspects of theinvention and is not intended to limit the scope of the invention.

SLE Patient Treatment Patient History

Patient RJ is a 30 year old African-American female who became ill witheasy bruising, fatigability and what was found to be thrombocytopenia.Upon failure of conventional therapy, she was splenectomized withimprovement of her thrombocytopenia.

The patient developed the symptomatology of SLE in 1993, and was foundto have thrombocytopenia, leukopenia, hypocomplementemia, elevatedanti-nuclear antibodies (ANA), and antibodies to double-stranded DNA(anti-dsDNA) above 1000. She developed severe vasculitis that was onlyminimally controlled with high doses of prednisone, azathioprine(IMURAN®) and various other immunoregulatory agents. In 1994, her bloodwas examined for evidence of what was thought to be Anaplasmataceae, andfound to have exogenous intra-erythrocytic bacterial structures thatwere morphologically indistinguishable from Anaplasma marginale.

Because of the severe disease of the patient, a trial of doxycycline wasbegun with express written consent of the institutional review board(IRB) and the patients's informed consent. The patient was begun ondoxycycline at a dose of 100 mg twice daily. She was instructed torefrain from meals containing calcium, magnesium or milk products whileon the medication, and was cautioned about the effects of sunlight onpatients with this antibiotic.

Doxycycline

The patient began subjective improvement within three weeks of therapy,and this continued until the doxycycline was discontinued after 15months. During this time she was able to increase her physical activityto walks of 3 to 5 blocks in the city. She noticed marked subjectiveimprovement but this still required her treatment with azathioprine, andprednisone in doses ranging from 20 to 60 mg daily. Severe oralexfoliative lesions appeared from time to time that were not related toHerpes or Candida infection as established by negative cultural andother diagnostic methods.

During this time, sequential examination of her blood showed a rapiddrop of parasitized erythrocytes, counted on the same microscope by acombination of phase contrast and careful giemsa staining. The drop inparasitized erythrocytes proceeded from more than 15 percent to lessthan 1 percent (about 0.7 percent) over one year, but plateaued at oneyear at approximately one percent. When the number of parasites began aslow rise to 1.1 percent in spite of continued therapy, concern beganfor the presence of developing resistance to the antibiotic.

Selection of Antibiotics

Because of the previously noted published activity of the thirdgeneration cephalosporins against intra-erythrocytic pathogens, theintra-erythrocytic location of the presumed pathogen, the likely slowmetabolic rate of cell division (deduced from the slow fall of parasitesunder doxycycline), and the protected intra-erythrocytic environment onthe organism, the following antibiotic combination was decided upon:

1. Cefpodoxime 400 mg twice daily with meals. Cefpodoxime is an orallyactive cephalosporin that has third generation activity. Cefpodoxime isonly 15 to 40 percent protein bound, and has activity against E. coliand Klebsiella at 1 μg. In addition, its serum activity after a 400 mgdose was 4 to 6 μg, and this could be doubled with concomitantprobenecid.

2. Probenecid at 500 mg twice daily. This was used to increase the bloodlevels to approximately twice their expected values.

3. Rifampin 300 mg twice daily. Rifampin was chosen because of itsability to penetrate cell walls, its oral dosage facility, and its knownsynergy with most antibiotics acting on cell walls.

The unidentified organism was presumed to have a peptidoglycan cell walland likely susceptibility to a third generation cephalosporin, as notedby Morrison et al., J. Clin. Microbiol., 24 (5):853-855 (1986).Intravenous therapy with any of the third generation cephalosporins for4 months would have the same effect, but because of that impracticality,the orally available cefpodoxime was chosen. Cefixime has similarcharacteristics and activities among the oral third generationcephalosporins, but is 90 percent protein bound, would presumably haveproblems traversing the erythrocyte membrane, and is less preferred.

Another consideration was the total number of organisms in thispatient's blood. At the time of initiation of therapy, 1.1 percent ofher erythrocytes had a parasite. The mode of activity of doxycycline isbacteriostatic, which presumes that one would wait for erythrocytesenescence and destruction for its elimination. Cephalosporins with orwithout rifampin are bacteriocidal, and there was concern that with theintracellular death of so many parasites, they would be expelled fromthe erythrocyte, (or the erythrocyte would degenerate), and thereticuloendothelial system would be instantly flooded with bacterialbodies, no longer shielded by erythrocyte membrane. The lesson of theJarisch-Herxheimer reaction in Syphilis appeared to have similarities.

Results of Therapy With Antibiotic Combination of Ceflodoxime andRifampin With Probenecid

The doxycycline was stopped on Dec. 23, 1995, and the above combinationtherapy was begun on the next day. On the eighth day of treatment, thepatient experienced chills, fever to 104° F., vomiting and diarrhea withdehydration that required her hospitalization. Her face suddenly becameflushed with a markedly lupus-like rash. Her stool was negative for C.difficile toxin, and she responded to intravenous therapy with fluids.Examination of her blood showed that the parasites had dropped to about0.7 percent from the previous about 1.1 percent. The antibiotictreatment was stopped on Dec. 31, 1995.

The patient thus experienced a reaction quite consistent with theJarisch-Herxheimer response or an allergic reaction to thecephalosporin. The latter allergic reaction was confirmed after arestart of the above three-drug antibiotic treatment caused anaccelerated reaction on Jan. 10, 1996. At that time about 1.4 percent ofthe patient's erythrocytes were parasitized. A Jarisch-Herxheimerreaction to the sudden release of bacterial parasites cannot becompletely ruled out.

The patient experienced a reaction quite consistent with an allergicdelayed hypersensitivity reaction, and on re-challenge appeared to havean accelerated reaction with continuous severe cutaneous burning. It isalso possible that she experienced a Jarisch-Herxheimer reaction,although this is considerably less likely. This change in parasitemiaindicated that approximately 40 percent of the intracellular parasiteshad been released or destroyed after 4-5 days of therapy withcefpodoxime and rifampin with probenecid.

The above 3-drug therapy was ceased and the patient was again treatedwith doxycycline on Jan. 12, 1996, after the level of exogenousbacterial structures (parasites) in her erythrocytes again climbed toabout 1.8 percent. After about one week on doxycycline, the percentageof parasitized erythrocytes declined to about 1.6 percent.

On Jan. 20, 1996, doxycycline treatment was stopped and the patientbegan treatment with clarithromycin at a daily dose of 1000 mg, withequal doses taken two times each day. Her erythrocytes were examinedmicroscopically as in FIGS. 1 and 2, and the percentage of parasitizederythrocytes was noted to decrease from about 1.6 to about 1.1 percentby Jan. 24, 1996, with some bacterial cell margins being indistinct atthat time. The presence of indistinct bacterial cell margins oftenindicates bacterial damage. No adverse reaction to the clarithromycinwas noted, and that treatment was continued with the percentage ofparasitized erythrocytes dropping to about 0.7 percent by Jan. 31, 1996.

The patient discontinued all antibiotic therapy in May of 1996. Whenexamined after two months, manifestations of Systemic LupusErythematosus had markedly increased after a previously prolonged periodof remission. The patient was very ill when examined in July of 1996,with constitutional symptoms of her disease, possibly exacerbated by anallergic reaction to the medications empirically administered for whatwas interpreted as cutaneous vasculitis. Her parasitemia had increasedto 1.3 percent from a previously recorded 0.6 percent andintra-erythrocytic parasites were readily visible by giemsa staining andphase contrast microscopy.

Clarithromycin was begun at a dose of 500 mg. twice daily, in July andafter one month of treatment, clinical remission of symptoms was almostcomplete. The level of parasitemia had dropped to 0.2 percent. Thislevel was rechecked for accuracy after two days and was verified ascorrect, and represented the lowest level of parasitemia ever observedin this patient.

In September 1996, rifampin at a dose of 300 mg twice daily wasadministered with the clarithromycin. Within three weeks, significantand marked changes had occurred in the visualized parasites with bloodof the patient as follows. Clinical improvement of the patient wasparallel to these changes 1) the numbers of previously visualizedparasites had fallen to 10 percent of their previous number, 2)approximately 70 percent of the parasites had changed when viewedmicroscopically: (a) some were fragmented, (b) some had changed theiroptical characteristics so that they were refractile under bright fieldmicroscopy, a characteristic which had never before been observed, and(c) under phase contrast optics, they changed to a larger refractileintra-erythrocytic structure unlike any had heretofore been observed.

All the changes seen strongly suggested bacterial dissolution and death.

Because a structure was still maintained within the erythrocyte, it wasprotected from the reticuloendothelial cells and was not absorbed aswould happen normally to an extra-cellular bacterial structure.

This patient had been continually treated palliatively with prednisonethroughout her illness. That prednisone dosage has been 20 mg per day.

These data support the etiologic role of bone marrow and erythrocyticparasitization by an unidentified bacterium in systemic lupuserythematosus, and the efficacy of macrolides such as clarithromycinwith a rifamycin derivative and/or a third generation cephalosporin witha rifamycin derivative in eliminating these organisms from theerythrocytes and bodies of patients with SLE and related conditions.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

I claim:
 1. A process for treating a patient with systemic lupus erythematosus whose erythrocytes contain exogenous bacterial structures stainable with acridine orange or giemsa and are visible in phase contrast microscopy that comprises repeatedly administering to said patient an antibacterial amount of a rifamycin along with an antibacterial amount of either a macrolide or a third generation cephalosporin in conjunction with an amount of probenecid that provides an adjuvant effect for said third generation cephalosporin.
 2. The process according to claim 1 wherein a macrolide is administered, and said macrolide is selected from the group consisting of erythromycin, clarithromycin, erythromycin 2'-acetate octadecanoate, erythromycin estolate and azithromycin.
 3. The process according to claim 1 wherein said rifamycin is rifampin or rifabutin.
 4. The process according to claim 1 wherein said third generation cephalosporin is selected from the group consisting of cefoperazone, cefmenoxime, cefteram, cefpimizole, cefodizime, cefixime, cefotaxome, cefsulodin, ceftazidime, ceftizoxime, ceftibuten, ceftiofur, ceftriaxone, cefuzonam, moxalactam and cefpodoxime.
 5. The process according to claim, 1 wherein said administrations are peroral.
 6. The process according to claim 5 wherein said third generation cephalosporin is cefpodoxime.
 7. The process according to claim 1 wherein said administrations are repeated periodically until said exogenous bacterial structures are eliminated from the patients erythrocytes.
 8. A process for treating a patient with systemic lupus erythematosus whose erythrocytes contain exogenous bacterial structures stainable with acridine orange or giemsa and are visible in phase contrast microscopy that comprises repeatedly administering to said patient an antibacterial amount of rifampin or rifabutin along with cefpodoxime in an antibacterial amount as well as an amount of probenecid that provides an adjuvant effect for said cefpodoxime.
 9. The process according to claim 8 wherein said amount of cefpodoxime administered is about 200 to about 800 mg per day, the amount of probenecid is about 250 to about 1000 mg per day and the amount of rifampin is about 450 mg to about 900 mg per day.
 10. A process for treating a patient with systemic lupus erythematosus whose erythrocytes contain exogenous bacterial structures stainable with acridine orange or giemsa and are visible in phase contrast microscopy that comprises periodically administering to said patient an antibacterial amount of clarithromycin plus an antibacterial amount of rifampin or rifabutin.
 11. The process according to claim 10 wherein about 250 to about 4000 mg of clarithromycin per day are administered to said patient with said antibacterial amount of rifampin or rifabutin. 